Sphingomonas abietis sp. nov., an Endophytic Bacterium Isolated from Korean Fir

PAMB 00755T, a bacterial strain, was isolated from Korean fir leaves. The strain exhibits yellow colonies and consists of Gram-negative, non-motile, short rods or ovoid-shaped cells. It displays optimal growth conditions at 20°C, 0% NaCl, and pH 6.0. Results of 16S rRNA gene-based phylogenetic analyses showed that strain PAMB 00755T was most closely related to Sphingomonas chungangi MAH-6T (97.7%) and Sphingomonas polyaromaticivorans B2-7T (97.4%), and ≤96.5% sequence similarity to other members of the genus Sphingomonas. The values of average nucleotide identity (79.9–81.3%), average amino acid identity (73.3–75.9%), and digital DNA–DNA hybridization (73.3–75.9%) were significantly lower than the threshold values for species boundaries; these overall genome-related indexes (OGRI) analyses indicated that the strain represents a novel species. Genomic analysis revealed that the strain has a 4.4-Mbp genome encoding 4,083 functional genes, while the DNA G+C content of the whole genome is 66.1%. The genome of strain PAMB 00755T showed a putative carotenoid biosynthetic cluster responsible for its antioxidant activity. The respiratory quinone was identified as ubiquinone 10 (Q-10), while the major fatty acids in the profile were identified as C18:1ω7c and/or C18:1ω6c (summed feature 8). The major polar lipids of strain PAMB 00755T were diphosphatidylglycerol, phosphatidylethanolamine, sphingoglycolipid, and phosphatidylcholine. Based on a comprehensive analysis of genomic, phenotypic, and chemotaxonomic characteristics, we proposed the name Sphingomonas abietis sp. nov. for this novel species, with PAMB 00755T as the type strain (= KCTC 92781T = GDMCC 1.3779T).

In this study, we isolated a new bacterial strain designated PAMB 00755 T from the leaves of Korean fir (Abies koreana) growing on Hallasan Mountain, Jeju Island, South Korea.In 2011, the International Union for Conservation of Nature (IUCN) identified a Korean fir (Abies koreana) as an endangered species in Korea.In recent years, to investigate the bacterial community associated with Korean fir and identify potential bioactive secondary metabolites of the host, we collected leaves from both healthy and diseased firs and analyzed the endophytic bacterial community [10,22,23].In this study, through a polyphasic taxonomic analysis of leaves from a healthy fir, we characterized strain PAMB 00755 T as a novel species in the genus Sphingomonas.

Isolation and Ecology
Leaves were collected from the Korean fir growing on Hallasan Mountain (33°2142N, 126°3145E) on Jeju Island, South Korea.To prepare the sample, five grams of leaves were subjected to surface sterilization using 1% NaOCl solution.The leaves were rinsed five times with distilled water.The sterilized leaves were then homogenized using a blender with 10 ml of 1× phosphate-buffered saline (PBS).To remove any debris, the resulting mixture was filtered through four layers of sterile cheesecloth and then serially diluted using a standard dilution method with 1× PBS buffer.A volume of 100 μl from the sample solution was evenly spread onto Reasoner's 2A agar (R2A, Difco) plates, followed by incubation at 25°C for 1 week.Single colonies were then re-streaked onto fresh R2A medium.Strain PAMB 00755 T , which formed circular, smooth, yellow colonies, was selected for the following study.The strain was stored at -80°C in 10% skimmed milk.It is currently accessible through the Korean Collection for Type Cultures (KCTC 92781 T ) and the Guangdong Microbial Culture Collection Center (GDMCC 1.3779 T ).Unless otherwise noted, bacterial cells were cultured on R2A for 3-4 days before performing subsequent tests.

16S rRNA Gene Sequence Analysis
The extraction of genomic DNA from the bacteria was performed to serve as a template for PCR amplification of the 16S rRNA gene.Universal bacterial primers, specifically 27F and 1492R, were used for the amplification process.The PCR products were sequenced by a commercial company (Macrogen Inc., Korea) using the primers 27F, 518F, 800R, and 1492R [24].The almost full length of the 16S rRNA sequence was assembled using Vector NTI software (1.6.1).The obtained sequences were compared in the EzBioCloud and the NCBI database [25].The alignment of multiple sequences was performed using BioEdit software.Subsequently, the construction of the phylogenetic tree was carried out using the Molecular Evolutionary Genetics Analysis (MEGA) software version 11.0.The neighbor-joining (NJ), maximum likelihood (ML), and minimum parsimony (MP) methods were used for this analysis, with 1,000 bootstrap iterations [26].The outgroup was Usitatibacter rugosus 0125-3 T .

Whole-Genome Characteristics
Genomic DNA was extracted as previously described [10].For whole-genome sequencing, the PacBio RSII system (Pacific Biosciences, Inc., USA) as well as an Illumina sequencing platform by Macrogen, Inc. (Korea), were utilized.The obtained raw data were de novo assembled using SMRT LINK (12.0) software.To evaluate the quality, completeness, and contamination of the assembled genome, the Microbial Genomes Atlas (MiGA) webserver was employed [27].The NCBI's Prokaryotic Genome Annotation Pipeline (PGAP) was utilized for genome annotation.To determine the overall genomic relatedness values between strain PAMB 00755 T and closely related strains, various tools were employed.The Genome-to-Genome Distance Calculation (GGDC) webserver (http://ggdc.dsmz.de/distcalc2.php) was used for digital DNA-DNA hybridization (dDDH) [28].The AAI calculator (http://enve-omics.ce.gatech.edu/aai/) was used for average amino acid identity (AAI) [29], and the ANI calculator (http://enve-omics.ce.gatech.edu/ani/) was used for average nucleotide identity (ANI).Based on the latest bacterial core gene set and pipeline (UBCG) analysis, a comprehensive phylogenomic tree was constructed.This analysis incorporated 92 core genes, ensuring a robust representation of the evolutionary relationships among the bacterial strains [30].The outgroup was Usitatibacter rugosus 0125-3 T .

Chemotaxonomic Characterization
Chemotaxonomic features, including cellular fatty acids, polyamines, isoprenoid quinones, and polar lipids, were investigated.For cellular fatty acid analysis, approximately 40 mg of fresh cells from the third quadrant of the streaked plates were collected.The cellular fatty acids were extracted as previously described [22] using a MIDI Sherlock Microbial Identification System (6.0).The samples were subjected to gas chromatography using a 6890N gas chromatography system (Agilent Technologies, USA), and the data were identified using the TSBA6 database of the Microbial Identification software package [31].Polyamines were extracted from 0.1 g of freeze-dried cells and analyzed by comparison to spermine, putrescine, spermidine, and homospermidine standards in Ace Emzyme Inc. (Korea).Isoprenoid quinones were extracted using the method outlined by Collins et al. (1980) [32] and subsequently analyzed by reverse-phase high-performance liquid chromatography.The extraction of polar lipids was carried out from 0.1 g of freeze-dried cells using a chloroform-methanol (2:1).Following the extraction, two-dimensional thin-layer chromatography was performed to separate the polar lipids that were obtained.To visualize the polar lipids on the TLC plates, four different spray reagents were used: molybdenum blue (Sigma-Aldrich, USA), 4% phosphomolybdic acid reagent, 0.2% ninhydrin (Sigma-Aldrich), and Dragendorff 's solution.

Phenotypic Characteristics
After culturing strain PAMB 00755 T on R2A medium for 3 days, the morphological features of the strain were examined using scanning electron microscopy (SEM) at the Korean Basic Science Institute in Chuncheon.To examine motility, a semi-solid R2A medium supplemented with 0.4% agar was used.The Gram staining procedure was conducted using a Gram staining kit (Difco), following the manufacturer's instructions.Catalase activity was assessed by observing bubbles production upon addition of a 3% (v/v) hydrogen peroxide solution to fresh cells.Oxidase activity was determined by observing the development of a purple color using an oxidase reagent kit.The strain was grown on various media, comprising R2A, nutrient agar (NA), potato dextrose agar (PDA), trypticase soy agar (TSA), marine agar 2216 (MA), and Luria-Bertani (LB) agar to determine the optimum medium.The strain was grown on PDA at 4, 10, 15, 20, 25, 30, 37, 40, 45, 50 and 60°C for 7 days to determine the optimal temperature.Salt tolerance was tested by growing the strain in R2A broth with concentrations from 0 to 15% (w/v) in increments of 1% [33], and pH tolerance was tested by growing the strain at a pH from 3.0 to 12.0 in increments of 1.Other biochemical features were tested using API 20NE (bioMérieux; substrate utilization, France) or API ZYM (bioMérieux; enzyme activities; NaCl 0.85% medium) and API 50CH (bioMérieux; production from carbohydrates) following the manufacturer's instructions.

Phenotypic, Physiological, and Biochemical Characteristics
Strain PAMB 00755 T exhibited optimum (and robust) growth on both PDA and R2A media, limited growth on NA, and no growth on TSA, LB, or MA.The cells are Gram-negative, non-motile, and short rods or ovoid (0.3-1.0 × 0.4-1.2μm) without flagella (Fig. S1).Colonies were yellowish and ranged from 2-4 mm in diameter after being grown for 3 days on PDA medium.The strain grew at pH 4.0-9.0(optimum pH, 6.0), 0-1% of NaCl (optimum NaCl concentration, 0%), and 4-25°C (optimum temperature, 20°C).It tested positive for catalase and oxidase activity.Other characteristics that distinguished strain PAMB 00755 T from closely related strains are shown in Table 1.

Phylogenetic Analyses
The complete 16S rRNA gene amplicon (1443 nucleotides) of strain PAMB 00755 T was obtained after assembly using Vector NTI software (1.6.1).Based on comparing the 16S rRNA sequence to the EzBioCloud and NCBI databases, strain PAMB 00755 T is related to members of the genus Sphingomonas, with the highest similarity to S. chungangi MAH-6 T (97.7%), S. polyaromaticivorans B2-7 T (97.4%), and S. oligoaromativorans SY-6 T (96.5%), and ≤96.0%similarity to other members of the genus Sphingomonas.In particular, the strain showed 93.0% 16S rRNA gene similarity to the recently proposed Sphingomonas nostoxanthinifaciens strain AK-PDB1-5 T based on using BioEdit software (7.2) [10].Based on the novel species recognition cut-off value of < 98.6% [34], the strain was classified as a novel species of the genus Sphingomonas.To establish the phylogenetic position of the strain, a phylogenetic tree was constructed using the 16S rRNA gene sequence.The results showed that strain PAMB 00755 T clustered with S. chungangi MAH-6 T and S. polyaromaticivorans B2-7 T (Fig. 1).Based on the similarity of the 16S rRNA gene sequence and phylogenetic tree analysis, strain PAMB 00755 T was finally identified as a member of the Sphingomonas.To perform comparative analysis under identical conditions, Sphingomonas.S. chungangi MAH-6 T (= KACC 19292 T ) and S. polyaromaticivorans B2-7 T (= KCTC 82794 T ) were selected.

Genomic and Phylogenomic Analysis
The complete genome of strain PAMB 00755 T was sequenced, resulting in a complete circular chromosome of 4,429,509 bp after de novo assembly using a microbial genome assembly application.The genome completeness, Table 1.Distinguishing physiological and biochemical characteristics of strain PAMB 00755 T and its closely related type strains.contamination, and quality values were determined to be 100, 0.9, and 95.5%, respectively.The G+C content of the genome was calculated to be 66.1%, similar to other species in the genus Sphingomonas, which have a high G+C content (57.4-70.5 mol%) (http://www.ncbi.nlm.gov/genome/?term=Sphingomonas).The genome annotation revealed the presence of 4,083 coding sequences (CDS), 59 tRNAs, and 12 rRNAs.Overall genome-related index (OGRI) analyses, involving dDDH, ANI, and AAI, were performed to determine the genetic relationships between strain PAMB 00755 T and closely related strains in the genus Sphingomonas.The dDDH values between strain PAMB 00755 T and its closely related strains of S. chungangi CGMCC 1.136 Considering the threshold values recommended for the bacterial species delineation, dDDH (<70%), ANI (<95-96%), and AAI (<95-96%) [35], strain PAMB 00755 T was identified as a novel strain in the genus Sphingomonas.The phylogenomic tree based on the whole genome also supported the placement of strain PAMB 00755 T within the genus Sphingomonas (Fig. 2).Fig. S2 shows a circular map of the strain PAMB 00755 T genome drawn using the CGView website.Annotation and analysis of secondary metabolite biosynthesis genes using anti-SMASH revealed that strain PAMB 00755 T contained a carotenoid biosynthesis cluster belonging to the terpenes (Table S1).The gene cluster involved in carotenoid biosynthesis could play an important role in antioxidant activity; however, further investigation is required to elucidate the exact bioactive chemicals and pathways for antioxidant activity.
Strain PAMB 00755 T is accessible from the Korea Collection for Type Culture (KCTC) with the designation KCTC 92781 T and the Guangdong Microbial Culture Collection Center (GDMCC) with the designation GDMCC 1.3779 T .The accession numbers for its 16S rRNA and whole-genome sequences are OP964609.1 and CP115174.1,respectively.Based on its distinct phenotypic, genotypic, and chemotaxonomic characteristics, strain PAMB 00755 T was classified as a novel species within the genus Sphingomonas.Therefore, we propose the name Sphingomonas abietis sp.nov.for this newly identified species.

Fig. 1 .
Fig. 1.Phylogenetic analysis of the 16S rRNA gene in strain PAMB 00755 T and related species within the genus Sphingomonas.The presented values indicate bootstrap values (>70%) obtained using the neighbor-joining (NJ), maximum likelihood (ML), and minimum parsimony (MP) algorithms.The scale bar represents 0.050 substitutions per nucleotide position.

Fig. 2 .
Fig. 2. Phylogenomic tree of genus Sphingomonas based on the up-to-date bacterial core gene set (UBCG) showing the position of strain PAMB 00755 T .At the nodes, the gene support index (GSI) is presented on the left side, while bootstrap values are indicated on the right side.The scale bar corresponds to 0.20 substitutions per position.

Table 2 . Comparisons of cellular fatty acid profiles between strain PAMB 00755 T and closely related strains in the genus Sphingomonas.
Sphingomonas abietis PAMB 00755 T ; 2, S. chungangi KACC 19292 T ; 3, S. polyaromaticivorans KCTC 82794 T ; "ND" represents not detected.The major components (>10%) are highlighted in bold.All the data presented in this study were obtained from the present study.a Summed features refer to groups of two or three fatty acids that cannot be individually distinguished using gas chromatography with the MIDI System.Summed feature 3 includes C 16:1 ω6c and/or C 16:1 ω7c, while summed feature 8 includes C 18:1 ω7c and/or C 18:1 ω6c.